Apparatus and methods for infrared calorimetric measurements

ABSTRACT

Apparatus and methods for performing calorimetry. The apparatus include optical devices for detecting thermal processes and multiwell sample plates for supporting samples for use with such optical devices. The methods include measurement strategies and data processing techniques for reducing noise in measurements of thermal processes. The apparatus and methods may be particularly suitable for extracting thermal data from small differential measurements made using an infrared camera and for monitoring chemical and physiological processes.

CROSS-REFERENCES

[0001] This application is a continuation-in-part of the following U.S.patent applications: Ser. No. 09/777,363, filed Feb. 5, 2001; Ser. No.09/777,368, filed Feb. 5, 2001; and Ser. No. 09/777,364, filed Feb. 5,2001. These applications, in turn, are continuations of U.S. patentapplication Ser. No. 09/764,963, filed Jan. 17, 2001, which claimspriority from the following U.S. provisional patent applications: SerialNo. 60/249,931, filed Nov. 17, 2000; and Serial No. 60/256,852, filedDec. 19, 2000. All of these patent applications are incorporated hereinby reference in their entirety for all purposes.

RELATED REFERENCES

[0002] This application incorporates by reference in their entirety forall purposes the following publications: Kenneth R. Castleman, DigitalImage Processing (1996); and Bob Sinclair, Everything's Great When ItSits on a Chip: A Bright Future for DNA Arrays, 13 THE SCIENTIST, May24, 1999, at 18.

FIELD OF THE INVENTION

[0003] The invention relates to calorimetry. More particularly, theinvention relates to apparatus and methods for performing calorimetrythat use optical devices to detect thermal processes and/or multiwellsample plates to support samples for use with such optical devices.

BACKGROUND OF THE INVENTION

[0004] Thermodynamics has established the interrelationship betweenvarious forms of energy, including heat and work. Moreover,thermodynamics has quantified this interrelationship, showing, forexample, that in chemical and physiological processes the differencebetween the energy of the products and the energy of the reactants isequal to the heat gained or lost by the system. In an “exothermic”process, this difference is negative, so that the process releases heatto the environment. Conversely, in an “endothermic” process, thisdifference is positive, so that the process absorbs heat from theenvironment. Thus, “calorimetry,” or the measurement of heat productionand/or heat transfer, can be used to determine if a chemical orphysiological process is exothermic or endothermic and to estimate theenergy produced or consumed.

[0005] The measurement of heat production and/or heat transfer inchemical and physiological processes can be quite complicated.Standardly, such measurements are made using a device known as a “bombcalorimeter.” This device typically includes a sturdy steel containerwith a tight lid, immersed in a water bath and provided with electricalleads to detonate a reaction of interest inside the calorimeter. Theheat evolved in the reaction is determined by measuring the increase intemperature of the water bath.

[0006] Unfortunately, bomb calorimeters are inadequate for themeasurement of heat production and/or heat transfer in many areas ofchemistry and physiology. For example, the study of processes involvinguncommon and/or expensive components may require analysis of samples toosmall for bomb calorimetry. Similarly, the high-throughput screening ofpharmaceutical drug candidate libraries for drug activity may requireanalysis of too many samples for bomb calorimetry.

[0007] The analysis of small samples is especially problematic due totheir small heat capacities and large surface-to-volume ratios. Manychemical and physiological processes lead to very small changes intemperature (<0.05° C.), making their analysis susceptible toenvironmental contamination. In particular, whenever there is atemperature difference between a sample and the environment, heat can beexchanged between the sample and the environment, for example, byconduction, convection, and/or radiation, among others. Such heatexchange may quickly alter the temperature of a small sample and therebyobscure any temperature change associated with a reaction. Moreover,fluid samples such as those typically used in studies of chemical andphysiological processes may initiate secondary reactions with theenvironment, such as evaporation. Evaporation, by definition, is anexchange of energy (moisture is added to the air, while chemical volumeis reduced). This process takes place on the surface of the sample,where the sample is exposed to the environment, and so may be especiallyproblematic for small samples due to their relatively largesurface-to-volume ratios. Evaporation not only removes energy from thesample, contaminating the measurement, but also may increase measurementnoise due to surface instability as the fluid phase changes to a gasphase.

SUMMARY OF THE INVENTION

[0008] The invention provides apparatus and methods for performingcalorimetry. The apparatus include optical devices for detecting thermalprocesses and multiwell sample plates for supporting samples for usewith such optical devices. The methods include measurement strategiesand data processing techniques for reducing noise in measurements ofthermal processes. The apparatus and methods may be particularlysuitable for extracting thermal data from small differentialmeasurements made using an infrared camera and for monitoring chemicaland physiological processes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a partially schematic cross-sectional view of a systemfor detecting thermal processes.

[0010]FIG. 2 is a top view of a multiwell sample holder for use with anoptical device for detecting thermal processes.

[0011]FIG. 3 is a cross-sectional view of the multiwell sample holder ofFIG. 2, taken generally alone line 3-3 in FIG. 2.

[0012]FIG. 4 is a graph showing the infrared transmissivity of apreferred sample well material as a function of the thickness of thematerial.

[0013]FIG. 5 is a pseudo-color image showing the extent and effect ofthermal cross talk in sample wells in (A) the microplate of FIGS. 2 and3 and (B) a standard commercial microplate.

[0014]FIG. 6 is a graph showing noise envelopes associated withmeasurements of sample temperature taken from the (A) top and (B) bottomof a sample well after application of common-mode noise subtraction,area averaging, and frame-averaging. The noise envelope associated withthe top-read data is significantly larger than the noise envelopeassociated with the bottom-read data due to evaporation.

[0015]FIG. 7 is a graph showing the size of the noise envelope as afunction of the number of frames average in an image-averagingexperiment.

[0016]FIG. 8 is a graph showing the effects of common-mode noise anddrift on thermal data collected using an infrared camera.

[0017]FIG. 9 is a graph showing the effects of offset subtraction onthermal data.

[0018]FIG. 10 is a graph showing the effects of removing common-modenoise on thermal data.

[0019]FIG. 11 is a software screen for use in the display of thermaldata, including temperatures and temperature differentials.

[0020]FIG. 12 is a software screen for use in the collection and/oranalysis of data from measurement and reference regions.

[0021]FIG. 13 is a software screen for use in defining selectedcharacteristics of the reference region.

DEFINITIONS

[0022] Technical terms used in this application have the meanings thatare commonly recognized by those skilled in the art. The following termsmay have additional meanings, as described below:

Common-Mode Noise

[0023] Typically low-frequency (<1 Hz) noise caused when internalcontrol loops, such as the servo on a cryogenic cooler, create responsechanges in the detector. In an infrared camera, these noise sources maybe common to each sensor element and may be geometrically displacedacross the sensor array. For example, at a given time, a thermal wavefrom the expansion of helium gas in a sensor cooler may cause slightgain changes in the sensor that cause a group of sensor elements in onegeometric region of the array to respond differently, or out of phasewith, another group of sensor elements in another region of the array.In most applications, common-mode noise is insignificant; however, inhigh-sensitivity (<0.05° C.) applications, common-mode noise may becomea limiting factor.

Heat

[0024] A form of energy associated with the motion of atoms ormolecules. Heat is capable of being transmitted by (1) conductionthrough solid and fluid media, (2) convection through fluid media, and(3) radiation through empty space.

Infrared (IR) Radiation

[0025] Invisible electromagnetic radiation having wavelengths from about700 nanometers, just longer than red in the visible spectrum, to about 1millimeter, just shorter than microwave radiation. Infrared radiationincludes (A) near IR (from about 700 nm to about 1,300 nm), (B) middleIR (from about 1,300 nm to about 3,000 nm), and (C) far or thermal IR(from about 3,000 nm to about 1 mm). Near and middle IR is infraredradiation that typically is caused by vibrations and low-levelelectronic transitions in molecules and that is only peripherallyrelated to heat. In contrast, thermal IR (or thermal infrared radiation)is infrared radiation that is caused or produced by heat and that isemitted by an object in proportion to the temperature and emissivity ofthe object.

Radiosity

[0026] The radiation emanating from an object is determined by thefollowing parameters: (1) emissivity, i.e., the amount of radiation theobject emits, (2) reflectivity, i.e., the amount of externally derivedradiation the object reflects, and (3) transmissivity, i.e., the amountof externally derived radiation the object transmits. For example, thethermal power P radiated by an object may be described by the equationP=εσAT⁴, where ε is the emissivity of the object, σ is theStefan-Boltzmann constant, A is the area of the object, and T is thetemperature. Emissivity, reflectivity, and transmissivity aredimensionless parameters with values that range between 0 and 1. For agiven material, the sum of these parameters should equal unity, so thateach parameter is inversely correlated with the sum of the otherparameters. A material with an opaque surface has a transmissivity ofzero, so its emissivity equals one minus its reflectivity. Materialsthat radiate very well and absorb a large percentage of the radiationthat strikes them have high emissivities.

Parasitic Noise

[0027] Typically low-frequency (<0.1 Hz) noise caused by stray radiationincident on the detector from within the detector housing, creating anoffset in output that results in measurement error. The stray radiationmay be caused by slight temperature changes internal and/or external tothe detector. In an infrared camera, the error may be geometricallydisplaced across the camera array, as determined by the efficiency ofthe cold shield for the camera sensor and the baffling within the Dewar.Most infrared cameras attempt to correct for parasitic noise using someform of internal calibration mechanism, such as a uniform-temperatureshutter that periodically drops in front of the sensor to perform anoffset compensation. These calibration mechanisms inherently interruptmeasurements and can lead to measurement errors if theuniform-temperature shutter is not actually perfectly uniform intemperature. All infrared radiometers have some form of parasitic noise.

Spatial Noise

[0028] Typically lower-frequency (<60 Hz) highly nonlinear noisereflecting detector artifacts caused by variations in the manufacturingprocess, for example, during metal oxide vapor deposition (MOVD). In aninfrared camera, these artifacts may cause slight differences in thegain or response characteristics, spectral characteristics, and/orstability characteristics of the various elements, columns, and/or rowsof the sensor. Spatial noise may result in low-frequency noise or adrift component, which may still remain even after performing acalibration for pixel gain and offset in the camera.

Temporal noise

[0029] Typically high-frequency (>60 Hz) random noise caused by(radiated or conducted) electronic noise, A/D quantization, 1/f noise,microphonics, and/or a low electronic signal-to-noise ratio from thedetector.

Thermal conductivity

[0030] The quantity of heat transmitted, due to a unit temperaturegradient, in unit time under steady conditions in a direction normal toa surface of unit area, when the heat transfer is dependent only on thetemperature gradient.

Thermodynamic Noise

[0031] Noise caused by thermodynamic instabilities in a medium, such asa fluid-to-gas phase transition. Surface measurements of most fluids,including water, show significant instability due to thermodynamic noisecaused by evaporation.

DETAILED DESCRIPTION

[0032] The invention provides apparatus and methods for performingcalorimetry (or thermogenic analysis). The apparatus include opticaldevices for detecting thermal processes and multiwell sample plates forsupporting samples for use with such optical devices. The methodsinclude measurement strategies and data processing techniques forreducing noise in measurements of thermal processes. The apparatus andmethods may be particularly suitable for (1) extracting thermal datafrom small differential measurements made using an infrared camera, and(2) for monitoring chemical and physiological processes.

[0033]FIG. 1 shows a system 100 for detecting thermal processes inaccordance with aspects of the invention. The system includes an opticaldevice 102 configured to detect thermal radiation 104 and a sample plate106 having a sample well 107 configured to support a sample 108 for usewith the optical device. The system may be used to monitor thermalprocesses in the sample or samples by detecting temperature changescorrelated with heat production (e.g., from a chemical or physiologicalreaction) and/or heat transfer in the samples. This correlation may beperformed using any suitable method, such as those described in thefollowing U.S. provisional patent application, which is incorporatedherein by reference: Serial No. 60/256,852, filed Dec. 19, 2000.Moreover, this correlation may be used to examine reactions, as well asmodulators such as agonists and/or inhibitors of the reactions. If thereare multiple samples, the radiation transmitted from the samples may bedetected sequentially from each sample, for example, by point reading,or simultaneously from some or all of the samples, for example, by imagereading. The optical device may include a detector 110 such as aninfrared optical sensor configured preferentially to detect thermalinfrared radiation. The detector measures thermal energy radiated from asample (or samples) supported by the sample plate and converts themeasured energy to a signal such as an electrical signal that can beconverted into a temperature, for example, using a blackbody or graybodyapproximation. In a preferred embodiment, the detector includes animaging device such as an infrared focal plane array (FPA) configured toobtain a time-dependent series of two-dimensional infrared images ofprocesses occurring in samples in a multiwell sample plate, permittingmeasurement of temperature and temperature changes in each process, as afunction of time, geometrically across the plate. The series of imagestypically is collected at a preselected frequency (typically>1 Hz) for apreselected period significant relative to a characteristic time of anytime-dependent process being monitored. The data subsequently may beprocessed and/or reported at the same and/or a lower frequency. The datamay be used to monitor, screen, rank, and/or otherwise analyze thermalprocesses occurring in the sample. The thermal analysis may be usedalone or together with other measurements to assess the presence,concentration, physical properties, and/or activity of a compound orcompounds in the sample. Thus, the system provides a noncontact,noninvasive method for measuring thermal properties such as temperature,in contrast to bomb calorimeters, thermometers, and capacitive andresistive circuits.

[0034] The system and its components may be configured to improve theaccuracy and/or sensitivity of thermal measurements, particularlythermal measurements involving small samples and/or small temperaturechanges. The optical device may be configured to reduce measurementerrors associated with noise, such as common-mode, parasitic, spatial,temporal, and/or thermodynamic noise, among others. The sample plate maybe configured to facilitate detection of thermal radiation through asurface of the plate and/or to reduce measurement-contaminating heattransfer between the samples and the environment (including othersamples). The optical device and sample plate may together be configuredto reduce noise associated with evaporation, for example, by using abottom-read detector and a sample plate having an infrared-transmissivebottom surface and in some cases a cover.

[0035] The remainder of the Detailed Description is divided into foursections: (A) optical devices, (B) noise reduction, (C) sample holders,and (D) examples.

A. Optical Devices

[0036] The optical device generally comprises any device capable ofpreferentially detecting thermal infrared radiation and using thedetected radiation to analyze thermal processes in a sample. The phrase“capable of preferentially detecting thermal infrared radiation” meansthat the device is configured and/or operated so that it detects morethermal infrared radiation than any other form of radiation (i.e., sothat at least about half of the radiation detected is thermal infraredradiation). The phrase excludes any device that detects thermal infraredradiation only incidentally, as might occur in an optical deviceconfigured to detect visible light if thermal radiation leaked into thedetector. The capability for preferentially detecting thermal infraredradiation may reflect use of one or more of the following mechanisms,among others: (A) use of spectral filters preferentially to “extract”thermal infrared radiating by blocking radiation other than thermalinfrared radiation, including visible, near IR, or middle IR radiation,(B) use of detectors having enhanced sensitivity for thermal infraredradiation, and/or (C) postprocessing of a detector signal to reduceand/or compensate for any component of the signal not resulting fromdetection of thermal infrared radiation.

[0037]FIG. 1 shows an optical device 102 constructed in accordance withaspects of the invention in use as a part of a system 100 for detectingthermal processes. The device includes a detector 110 configured todetect thermal infrared radiation emitted by a sample, a stage 112configured to support a sample in a sample holder for thermal analysisby the detector, and a processor 114 configured to analyze radiationdetected by the detector. The detector and stage are positioned suchthat at least a portion of the thermal infrared radiation emitted by thesample is incident, indirectly or preferably directly, on the detector.This may be accomplished by ensuring that a central axis CA of thesample wells is aligned with an optical axis OA of the instrument priorto detection of thermal infrared radiation. Alignment simply means thatthe two axes are sufficiently close to parallel that radiation from acentral portion of the sample well is detectable by the instrument. Forexample, in FIG. 1, the central axis of each sample well is aligned withthe optical axis of the instrument. The detector may be positioned belowthe stage to form a “bottom-read” instrument, above the stage to form a“top-read” instrument, or in other positions to form other instruments.The instrument of FIG. 1 is a bottom-read instrument, and the instrumentof FIG. 1 with components of the optical device 102 inverted andpositioned above the stage is a top-read instrument.

[0038] The stage may be movable, so that samples may be deposited at afirst position, moved to a second position for fluid dispensing, movedto a third position for thermal equilibration, moved to a fourthposition for thermal detection, and moved to a fifth position forpickup. These positions may be the same or different, and any givenposition (except the detection position) may be present or absent. Thestage may move sample holders translationally and/or rotationally, amongothers.

[0039] The sample generally comprises any object or system of objectsintended for thermal analysis. The sample may include compounds,mixtures, surfaces, solutions, emulsions, suspensions, cell cultures,fermentation cultures, cells, tissues, secretions, and/or derivativesand/or extracts thereof. The sample also may include the contents of asingle sample site, or several sample sites, depending on the assay.

[0040] The detector generally comprises any device for preferentiallydetecting thermal infrared radiation and converting the detectedradiation into a signal representative of the detected radiation. Apreferred detector is an imaging detector, such as an infrared camera,that is capable of simultaneously viewing part or all of a sampleholder. Further aspects of the detector are described below in SectionB.

[0041] The stage generally comprises any mechanism for supporting asample in a sample holder at an examination site for thermal analysis bythe device. A preferred stage is a transporter capable of moving thesample holder vertically and/or horizontally between the examinationsite and one or more transfer sites where the sample holder can beloaded onto and/or unloaded from the stage.

[0042] The processor generally comprises any mechanism for analyzing thesignal from the detector, for example, to conduct a thermal analysis.The analysis may include conversion of a signal representative ofintensity and/or wavelength into a signal representative of temperatureand/or differential temperature, among others. The analysis also mayinclude performing calculations to reduce noise and/or to facilitatedata reporting, as described below. The processor may be intrinsic tothe detector, extrinsic to the detector, or both. Further aspects of theprocessor are described below in Section B.

[0043] The optical device also may include a housing 116 to support andprotect the detector. The housing may include, among others, an opticstube 118, an infrared-transmissive window 120, and/or a baffle 122having an aperture 124. The optics tube may support the detector and/orcomponents of the housing, such as the window and baffles. The opticstube also may reduce the amount of unintended thermal infrared radiationentering the detector. The window may permit thermal infrared radiationto enter the housing for detection, while also permitting the housing tobe sealed to reduce contamination and/or (partially) evacuated to reduceabsorption and/or scattering of thermal radiation prior to detection.The window may include a portion formed of zinc selenide (ZnSe) and/orpolyethylene, among others. The baffles may block stray thermal infraredradiation. The optical device may be configured to shield the samplefrom incident radiation to reduce the proportion of the sample signalarising from transmission, reflection, and/or photoluminescence from thesample.

[0044] The optical device also may include a data output mechanism suchas a view screen or printer for reporting results of any thermalanalysis. Data generally may be reported using any suitable method,physical and/or electronic, digital and/or analog, and static and/ortime varying, among others. Suitable methods include tables, graphs,and/or images, among others. Data may include temperatures and/ortemperature differentials, among others, at a fixed time or as afunction of time. Further aspects of the data output mechanism includingsuitable software are described below in Section D.

B. Noise Reduction

[0045] Noise is almost invariably a problem in measurements of thermalprocesses. However, noise may be especially problematic in measurementsof thermal processes involving small samples and/or small temperaturechanges, where even minor noise can obscure or overwhelm any temperaturechange associated with the thermal process.

[0046] The effects of noise on measurements of thermal processes can bereduced using noise reduction techniques. Noise reduction involvesidentifying sources of noise (e.g., measurement noise, camera noise,etc.), and then developing methods for reducing or eliminating the noiseand/or its effects. Unfortunately, thermal detectors such as infraredcameras are susceptible to several types of noise, includingcommon-mode, parasitic, spatial, and/or temporal noise, among others. Inaddition, fluid samples are susceptible to other types of noise,including thermodynamic noise, which is caused by thermodynamictransformations, such as evaporation, in which the sample changes from afluid to a gas. These and other forms of noise are described above,under Definitions. Thus, noise reduction in measurements of thermalprocesses may be facilitated by the application of one or more differentmethods, and/or by the repeated application of the same and/or relatedmethods.

[0047] The present “state of the art” for infrared, radiometric camerasdefines measurement performance in terms of noise-related parameters,specifically, accuracy and sensitivity. Here, accuracy is a relativeabsence of error or mistake, and sensitivity is an ability to detect ormeasure an input, especially a weak input. The sensitivity of infraredcameras normally is quantified in terms of noise equivalent temperaturedifference (NETD), which is the RMS noise/response at a giventemperature, f#, and operating frequency (normally 30° C., f/1, and 60Hz). NETD typically is the limiting factor in determining measurementsensitivity. A typical, state-of-the-art, high-performance infraredcamera, such as the FLIR SC3000, using Quantum Well (QWIP) sensortechnology, specifies an absolute accuracy of about 2° C. and asensitivity (NETD) of about 0.03° C. Unfortunately, this sensitivity maybe inadequate in measurements of small temperature changes.

[0048] The invention provides methods for reducing noise and/orenhancing accuracy and/or sensitivity in thermal measurements,particularly measurements involving small samples and/or smalltemperature changes. These methods may improve upon the previousstate-of-the-art sensitivity described above, potentially providingsensitivities of <0.1° C. or even <0.01° C. and RMS noise levels of<0.05° C. or even <0.005° C., at least when the methods are used on datacollected with preferred sample holders.

[0049] The methods may be implemented using any suitable apparatus, suchas those described above in Section A. These apparatus may include oneor more processors associated intrinsically and/or extrinsically withthe optical device and incorporating instructions for and capable ofcarrying out the functions specified by the methods. The processor(s)may perform during signal acquisition by the optical device, forexample, so that the most recent image is a processed image formed as arunning average of the actual most recent image and a preselected numberof preceding images. Alternatively, or in addition, the processor(s) mayperform after signal acquisition, for example, in a central processingunit used to run the apparatus and/or experiments conducted using theapparatus.

[0050] The methods may be performed using any suitable analysis mode,including continuous (analog) and/or discrete (digital) modes, andpoint-reading and/or imaging modes. Thus, the signal may be collectedusing an analog camera (such as a video camera) and/or a digital camera(such as a charge-coupled device (CCD) camera), and/or using an analogand/or a photon-counting point-reading detector (such as aphotomultiplier tube, photodiode, etc.). In an exemplary embodiment,signals are collected in imaging mode, and analog signals (if any) areconverted to digital signals prior to and/or during analysis. A digitalsignal typically takes the form of one or more digital images, whichtypically comprise two- or three-dimensional quantized functions thathave been generated by optical means, sampled using a defined spatialpattern (such as an equally spaced rectangular grid), and representedusing a defined presentation scale (such as an equal level gray scale ora pseudocolor scale). A two-dimensional digital image T may berepresented as an M×N matrix of values T_(ij) associated with spatialpositions i and j. A series of two-dimensional digital images (such as atime series) may be represented as an M×N×k matrix of values T_(ij)(k)by further associating a time index k with each spatial value T_(ij).

[0051] The methods may involve point operations and/or area operations,among others. Here, a point operation is an operation that modifies dataon a point-by-point (e.g., pixel-by-pixel) basis depending only on thevalue and/or location of the data point. In contrast, an area operationis an operation that modifies data on a point-by-point basis dependingat least in part on the value and/or location of points in theneighborhood of the data point. The methods may involve replacing agiven number of data points or images with an equal number ofpotentially modified data points or images. Alternatively, or inaddition, the methods may involve replacing two or more data points orimages with a smaller number of data points or images.

[0052] The invention may involve application of one or more of thefollowing techniques, among others, as described below: (1) low-passfiltering, (2) temporal averaging, (3) spatial averaging, (4) referencecalibration, (5) offset subtraction, and/or (6) bottom reading.

[0053] 1. Low-pass filtering

[0054] Low-pass filtering generally comprises any method for reducing oreliminating high-frequency temporal and/or spatial noise by blocking orotherwise suppressing frequencies above a certain “cutoff frequency”while passing or otherwise promoting frequencies below the cutofffrequency. Thus, to reduce high-frequency “temporal noise” in thermalimages, data may be collected at a relatively high frame rate and passedthrough a low-pass time-domain filter to cut out the high-frequencynoise. In an exemplary embodiment, full-field radiometric data arecollected using an infrared camera as described above at a 60-Hz framerate and passed through a low-pass filter internal to the cameraelectronics to yield filtered data corresponding to a lower frame rate.Suitable low-pass filters are described in Kenneth R. Castleman, DigitalImage Processing (1996), which is incorporated herein by reference. Inparticular, suitable low-pass filters include (1) simple low-passfilters, such as the box filter, triangular filter, and high-frequencycutoff filter, and (2) more complex low-pass filters, such as theGaussian low-pass filter. A preferred low-pass filter includes aframe-average as described below in which the most recent imagerepresents a frame-average of the most recent image with one, three,seven, or fifteen of the most recent preceding images, among others.

[0055] 2. Temporal averaging

[0056] Temporal averaging generally comprises any method for summarizingor representing the general significance of a set of unequal values of aquantity or function sampled at different times, such as determinationof a mean, mode, or median value, among others. In an exemplaryembodiment, temporal averaging involves application of frame-averaging,among others. Here, frame-averaging may be used to reduce any residualhigh-frequency temporal noise remaining after application of a low-passfilter.

[0057] Frame-averaging generally comprises averaging (or otherwisesmoothing) data over a series of frames. For example, a pixelatedfunction T may be frame-averaged over N frames by summing the product ofthe value of the function T_(ij) and a corresponding weighting factorW_(ij) in each frame k, and then dividing the sum by the sum of theweighting factors: $\begin{matrix}{{\langle T_{ij}\rangle}_{FA} = {\frac{\sum\limits_{k = 1}^{N}{{W_{ij}(k)}{T_{ij}(k)}}}{\sum\limits_{k = 1}^{N}{W_{ij}(k)}}\quad \overset{\quad {W_{ij} = {1\forall_{{ij}\quad}}}}{\rightarrow}\quad {\frac{1}{N}{\sum\limits_{k = 1}^{N}{T_{ij}(k)}}}}} & (1)\end{matrix}$

[0058] Here,

_(FA) denotes frame-averaging. The weighting factors all may be equal(for example, they all may be equal to one), or some or all of theweighting factors may have different values, depending on the algorithm.The frame-averaged frame may be used to replace an input frame,typically a middle or final frame, in the series of N frames used toconstruct the average. The number of frames will be unchanged (absentedge effects) if each frame in a series of frames is replaced with aframe-averaged frame. In contrast, the number of frames will be reducedif two or more frames are replaced with a single frame-averaged frame.

[0059] The preferred number N of frames to use in the frame-average isdetermined by competing factors. Generally, it is better to use a largernumber of frames because frame-averaging typically reduceshigh-frequency random temporal noise by the square root of the number offrames averaged. However, the overall time corresponding to the numberof frames used in the average should be small relative to the time scaleof thermal changes in the system to avoid averaging frames that differdue to actual differences in the temperature of the sample rather thandue merely to noise. In the data presented below in Section D, theoptimum number of frames for frame-averaging was between about 4 and 16.

[0060] Frame-averaging may be performed simultaneously and/orsequentially for different samples, and/or for measurement and referenceareas.

[0061] 3. Spatial averaging

[0062] Spatial averaging generally comprises any method for summarizingor representing the general significance of a set of unequal values of aquantity or function sampled at different positions, such asdetermination of a mean, mode, or median value, among others. Data maybe spatially averaged to return a reduced number of (average) values ora single (average) value for each area as a function of time. Forexample, a pixelated function T may be area-averaged for a particularframe k by summing over some or all of the M pixels ij in a given area Aof a frame the product of the value of the function T_(ij) and acorresponding weighting factor W_(ij), and then dividing the sum by thesum of the weight factors: $\begin{matrix}{{{\langle T\rangle}_{AA}(k)} = {\frac{\sum\limits_{i,{j \in A}}{{W_{ij}(k)}{T_{ij}(k)}}}{\sum\limits_{i,{j \in A}}{W_{ij}(k)}}\quad \overset{\quad {{W_{ij}{(k)}} = {1\forall_{ij}}}\quad}{\rightarrow}\quad {\frac{1}{M}{\sum\limits_{i,{j \in A}}{T_{ij}(k)}}}}} & (2)\end{matrix}$

[0063] Here,

T

_(AA) denotes area-averaging. The weighting factors all may be equal(for example, they all may be equal to one), or some or all of theweighting factors may have different values, depending on the algorithm.Thus, in a sample holder having 96 sample wells each having ameasurement area and a reference area, area averaging may be used toreduce the data set to as few as 96 measurement values and 96 referencevalues by independently averaging pixel values over all or part of eachmeasurement and reference area. The measurement and reference areas maybe distinguished using application software implemented in theprocessor. Area averaging typically involves >4 pixel elements andpreferably involves >9 pixel elements. Area averaging may reduce theeffects of geometric, spatial noise common to most FPA detectors. Suchnoise may reflect defective or nonlinear detector elements and/or slightdifferences in amplifier characteristics. In some applications, areaaverages may be computed as median values or mode values, asappropriate, instead of mean values.

[0064] The following table shows an exemplary algorithm for areaaveraging data from contiguous measurement and reference regions. Thenumber of data points used in the average and the number of averagesreported by region may be selected according to any criterion orcriteria. Generally, most or all of the data points unambiguouslyascribable to one or the other region but not both will be used in theaverage, and, generally, one average will be reported for each region.

[0065] 4. Reference calibration

[0066] Data may be calibrated using a reference standard, such as anadjacent local (e.g., perimeter) reference standard, for example, bysubtracting a reference value from a corresponding measurement value toreturn a differential measurement for each sample well as a function oftime.

T _(RC) =T _(Meas) −T _(Ref)   (3)

[0067] Here, the measured and reference values may be properties of thethermal radiation detected from the measurement and reference regions,respectively, such as intensities, or they may be quantifies derivedfrom such properties, such as temperatures. The method may be appliedpixel-by-pixel or area-by-area, among others. Subtracting referencevalues from measurement values may reduce or eliminate common-modenoise, internal drift, and/or parasitic noise local to the region of thedetector array used in the measurements. These noise sources have atendency to be geometrically dispersed across the sample holder orsample wells, so that other noise-reduction techniques, such assingle-point reference or Fourier transform characterization andsubtraction have limited success. These other methods have a tendency toamplify noise where it shifts out of phase relative to adjacent areas,whereas the local reference compensates for geometric shifting.

[0068] 5. Offset subtraction

[0069] Data may be adjusted by subtracting one or more offsets from eachmeasurement.

T _(OS) =T−T _(Offset)   (4)

[0070] The offset may be used to set the initial-time differentialmeasurements for each sample well at t (time)=0, so that there is acommon starting point from which to measure changes in temperature.Offset subtraction effectively creates a zero reference at the beginningof the experiment and adjusts the difference in temperature between themeasurement region and associated reference region to zero. Adjustingthe offset to zero may compensate for field nonuniformity resulting fromcamera drift prior to the start of data collection.

[0071] In some applications, data may be adjusted further bymultiplication (or, equivalently, division) by a suitable scalingfactor.

[0072] 6. Bottom reading

[0073] Reading through the bottom of an infrared-transmissive samplewell may reduce thermodynamic noise created at the interface of dry airand the sample. In particular, evaporation at sample surfaces exposed todry air may create a saturated gas layer adjacent the sample surface.This layer may be opaque or nearly opaque to the thermal detector andshow significant instability (measured to be >0.05° C.). Measurementnoise created by evaporation may be fivefold or more greater thanmeasurement noise associated with reading through the bottom of thesample well or from an independent black body reference. Additionally,evaporation at the surface may lower the surface temperatures measuredby the camera by as much as 2° C. This 2° C. difference is a heat sinkfor the reaction being measured. Bottom reading allows the top surfaceof the sample well to be sealed so that the space above the samplebecomes saturated with moisture, reducing evaporation noise and heatloss.

[0074] The application of these noise-reduction methods generally isquite flexible. For example, each method generally may be appliedseparately, alone or in combination with any number of other methods.Moreover, each method generally may be applied in any order.

C. Sample Holders

[0075] The sample holder (or, equivalently, the sample substrate orsample plate) generally comprises any substrate or material capable ofsupporting a sample or samples for thermal analysis. The sample holderpreferably is capable of supporting a plurality of samples at discrete(i.e., individually distinct) sample sites. Suitable sample holders mayinclude microplates, PCR plates, microarrays, chromatography plates, andmicroscope slides, among others, where microplate wells and biochiparray sites comprise assay or measurement sites. Exemplary microplatesare described below in detail. These and similar sample holders such asPCR plates generally keep each portion of each sample from mixing witheach portion of each other sample. Exemplary microarrays (or,equivalently, biochips) are described in detail in Bob Sinclair,Everything's Great When It Sits on a Chip: A Bright Future for DNAArrays, 13 THE SCIENTIST, May 24, 1999, at 18. These and similar sampleholders generally keep at least a portion of each sample from mixingwith at least a portion of each other sample, for example, by adheringthe molecules of interest such as nucleic acids or proteins to specificarray sites while allowing the solvent and any associated dissolved orsuspended species to move between sites.

[0076] The sample holder may include a thermal isolation structuredisposed between the sample wells to reduce thermal transfer between thewells and the environment and thus between adjacent wells. The thermalisolation structure may include a thermal buffer, thermal barrier,and/or isolation well, among others, as described below. The thermalisolation structure may be composed at least in part of a differentmaterial than the sample wells. The thermal isolation structure maysubstantially surround a central or optical axis of each sample well,isolating the wells without obstructing transmission of thermal infraredradiation along the central axis. The thermal isolation structure alsomay be disposed such that any straight line below a plane formed by thetops of the sample wells connecting a portion of one sample well to aportion of an adjacent sample well intersects the isolation structure.

[0077] The sample holder also may include an insert member defining anarray of sample wells and a support member having a thermal isolationframework in a configuration corresponding to the array of sample wells.The sample wells each may have a central axis, and the insert may engagethe support member such that each sample well is thermally isolated fromadjacent sample wells without obstructing the transmission of thermalinfrared radiation along the central axis. The thermal isolationframework may include a thermal buffer, thermal barrier, and/orisolation well, among others, as described below.

[0078] The sample holder also may include an insert having a pluralityof sample wells, and a thermal isolation member for supporting theinsert so that each sample well can be precisely positioned along anoptical path, where the thermal isolation member provides a thermallycontrolled thermal reference surface adjacent each well as viewed alongthe optical path. The reference surface may define an aperture thatframes the associated optical path.

[0079] A preferred sample holder is configured as a microplate having aframe and a plurality of sample wells disposed in the frame for holdinga corresponding plurality of samples for analysis. This format maycombine small-volume samples and a high-density holder, permittingautomated analysis of large numbers of samples. This format also may beconfigured to reduce unintended heat exchange between the samples andthe environment (including between the sample and other samples) and/orto permit an optical detector to measure thermal infrared radiationtransmitted through a surface of the sample holder.

[0080] The sample holder may include one or more of the followingfeatures, among others:

[0081] 1. Thin surface

[0082] A sample well having at least one surface having a thickness ofless than about 0.005 inches, and preferably less than about 0.001inches, and most preferably less than about 0.0005 inches. A thinsurface may be important for at least two reasons: (1) increasedinfrared transmissivity, and (2) decreased thermal conductivity. Thesetwo criteria preferably may be met using a single material, such as apolymeric polyethylene blend having a high infrared transmissivity(e.g., greater than about 50% or about 80%) and a low thermalconductivity (e.g., less than about 1 W/m-K or about 0.6 W/m-K).

[0083] A thin (i.e., reduced-thickness) surface may increasetransmissivity. A thin surface may be less likely to absorb thermalenergy being radiated by the sample due to its shorter path length andmore likely to have an outer (i.e., non-sample-contacting) surface atthe same temperature as the sample, facilitating calorimetric analysisthrough the surface. A preferred thin surface has a high transmissivity(e.g., >80%) for thermal infrared radiation, particularly thermalinfrared radiation having wavelengths between about 3 and 5 micrometersand between about 7 and 14 micrometers. (These wavelength ranges may beespecially useful in thermal imaging, because they correspond to minimain atmospheric absorption.) A thin more transmissive surface preferablyis located at least at the bottom of the sample well to permit detectionfrom the underside of the sample holder using a bottom-read analyzer.The surface may be substantially (e.g., optically) flat to reduceoptical aberrations during analysis through the surface.

[0084] A thin (i.e., reduced-thickness) surface also may decreasethermal conductivity. There are three primary mechanisms for heattransfer in the plate: conduction, convection, and radiation. Typically,conduction is the most significant mechanism, and radiation is the leastsignificant mechanism. Conduction may be described by the equationP=KA∇T, where K is the thermal conductivity, A is the surface area, and∇T is the temperature gradient. Thus, reducing surface area may reduceconduction. A primary path for conduction is through the walls of thesample well to contact points on the associated frame. This path may bereduced using thin-walled sample wells. Moreover, because the thermalconductivity of air (˜0.02 W/m-K) is less than that of the preferredwell material (0.6 W/m-K), it is important to use the air as much aspossible for a conduction path. Thus, the wells hold heat much like athermos. Finally, the thermal capacitance of a thin material is lower,so that there is less change in temperature due to the initial ΔT in thesystem. In particular, the material may be selected such that thethermal mass of the sample wells is no more than about half the thermalmass of an aqueous sample positioned in the sample well, even when thesample well is completely full. A thin less conductive surfacepreferably is located at least at the sides of the sample well.

[0085] 2. Thermal buffer

[0086] A thermal buffer disposed between the sample wells to resistthermal transfer between sample wells, or between the environment andthe sample wells. The thermal buffer generally comprises any mechanismfor resisting a change in temperature. In this sense, the thermal bufferresembles a pH buffer, which resists a change in pH when an acid or baseis added to a solution by binding to the added species, or an electricalcapacitor, which resists a change in voltage by storing or releasingcharge. The thermal buffer may be used to buffer (or keep relativelyconstant) the temperature of any structure adjacent the sample well,such as the trapped volume described below. The thermal buffer mayinclude a structure having a high thermal mass (or heat capacity), whichcan absorb heat without undergoing a significant change in temperature.This high thermal mass structure may, for example, have a substantiallyhigher thermal mass (or heat capacity) than the sample wells and/orcorresponding samples, for example, three, five, or even ten timeshigher. The high thermal mass structure may include a metal such asaluminum and/or a high thermal capacitance plastic, among others.

[0087] 3. Thermal barrier

[0088] A thermal barrier disposed between the sample wells to blockthermal transfer between sample wells. The thermal barrier generallycomprises any mechanism for blocking the transfer of heat into or out ofthe sample wells or the vicinity of the sample wells, such as anadjacent trapped volume. The thermal barrier may include a materialhaving a low emissivity and/or a high reflectivity for infraredradiation. For example, the thermal barrier may include a material thatreflects at least about half of the infrared radiation that otherwisewould be incident upon surfaces of the sample well. Generally,emissivity and reflectivity are inversely related; thus, shiny, metallicmaterials tend to have low emissivities and high reflectivities, whereasmatte, dark-colored materials tend to have high emissivities and lowreflectivities. In a preferred embodiment, the thermal barrier includesa material having a reflectivity of at least about 0.8 and an emissivityof at most about 0.2.

[0089] 4. Double-walled sample wells

[0090] A double-walled sample well, formed, for example, by positioninga sample well in a corresponding isolation well. In a preferredembodiment, a plurality of isolation wells are disposed in a frame, acorresponding plurality of sample wells are disposed in the isolationwells, and none of the sample or isolation wells is in fluid contactwith another of the sample or isolation wells. The double-walled wellsmay include a trapped volume formed between an outer surface of thesample wells and an inner surface of the corresponding isolation wells,further reducing thermal transfer to and from samples positioned in thesample wells. The trapped volume may enclose air and/or an inert gas,and/or be partially or fully evacuated relative to standard atmosphericpressure. The trapped volume also may enclose or be lined along itsperimeter with a thermal barrier, i.e., a material having a lowemissivity and/or a high reflectivity for infrared radiation to reduceradiation thermal transfer to and from the sample well.

[0091] 5. Plural optically transmissive surfaces

[0092] A plurality of optically transmissive surfaces, at least oneassociated with the frame and at least one associated with the samplewell, where the surfaces are configured so that an optical reader candetect electromagnetic radiation such as infrared radiation transmittedfrom a sample through both the optically transmissive surface of thecorresponding sample well and the optically transmissive surface of theframe. For example, a plurality of optically transmissive surfaces maybe formed by corresponding surfaces of a sample well and isolation wellin a double-walled well, as described above.

[0093] 6. Measurement and reference regions

[0094] A combination of a measurement region and a reference region. Themeasurement region may be a portion of a sample well, and the referenceregion may be an adjacent portion of the frame, isolated from the samplewell, particularly a high-thermal-mass and/or high emissivity (>0.5 andpreferably >0.8) surface portion capable of acting as a blackbody orgraybody reference. The reference region may be composed at least inpart of a different material than the sample wells and may include ametal such as aluminum. The reference region(s) may be disposed adjacent(e.g., about or between) the sample wells, so that each measurementregion is near a corresponding reference region, reducing artifacts thatreflect temperature drift across the sample plate. Thus, an M×N array ofmeasurement regions might be complemented by an M×N of reference regionsdisposed about the measurement regions, or an (M−1)×(N−1) array ofreference regions disposed between the measurement regions. Thereference region may be configured as a ring or annulus distributedabout or adjacent a perimeter of the sample well and/or about andpreferably symmetrically about a central axis of the sample well. Thereference region may be positioned about or above the top of acorresponding sample well, and/or about or below a corresponding samplewell. The reference regions and the corresponding sample wells may beseparated by a gap such as an air gap along a line connecting eachportion of the thermal reference regions and the corresponding samplewells to reduce heat transfer between the sample wells and the thermalreference regions. Thermal characteristics of the measurement region maybe calibrated using thermal characteristics of the reference region, forexample, by subtracting the reference characteristic from themeasurement characteristic. This calibration may reduce geometricallydispersed common-mode noise, including the effects of internal parasiticradiation and camera drift. The use of dedicated reference regions mayfree up all of the sample wells for data analysis, because none of thewells needs to be used as a reference well.

[0095] 7. Consumable sample well inserts

[0096] A combination of a reusable frame and a consumable sample wellinsert (or a set of consumable sample well inserts) configured to fitwithin or mate with the frame. The combination may facilitate reuse ofportions of the sample holder that are expensive, such as the thermalbuffer and/or thermal barrier. The combination also may facilitatedisposal of portions of the sample holder that contact the sample byreducing the amount of such materials that must be discarded. Thecombination may be constructed so that the insert is substantiallysupported by the frame yet substantially thermally insulated or isolatedfrom the frame. The frame and the sample wells may be composed of thesame or preferably different materials. Here, consumable may be definedas more likely to be discarded than reused, typically because it is moreconvenient and/or less expensive to be discarded than reused. Forexample, a consumable sample well insert may obviate the need to cleansample wells between samples.

[0097] 8. Cover

[0098] A cover configured for use with the sample holder. The covergenerally comprises any mechanism for covering the sample holder, or aportion of the sample holder, to reduce contamination of the samplesand/or to reduce evaporation from the samples (e.g., by reducingexposure to dry air and/or convective air currents), among others. Thecover generally may be formed of any suitable material, such as a rigidplastic and/or a thin layer of oil or other less evaporative materiallayered over the sample. The cover may be infrared transmissive, so thatsamples may be analyzed through the cover using a top-read analyzer. Thecover may be configured to touch the top surface of the sample.Alternatively, the cover may be configured to leave an air gap betweenthe top surface and the cover, particularly a small air gap that mayquickly saturate with fluid vapor after fluid samples are positioned inthe wells and before reactant or catalyst are delivered to reduceevaporative cooling during analysis. Generally, evaporation may bereduced by reducing the size of the air gap, for example, by usingshallow wells and/or by substantially filling the wells (for example,until the samples occupy at least about half or even about eight-tenthsor nine-tenths of the volume of the sample wells). Alternatively, or inaddition, evaporation may be reduced by increasing the humidity of theair adjacent the sample well or sample holder. The cover may include anaperture so that a fluid delivery system such as a pipette can piercethe cover and deliver reactant fluids.

D. Examples

[0099] The following examples describe without limitation furtheraspects of the invention. These examples show that thermal cross talkbetween sample wells can be reduced by thermally isolating the samplewells and that thermal resolution and the accuracy and sensitivity ofthermal measurements can be enhanced by reducing noise, includingcommon-mode, parasitic, spatial, temporal, and/or thermodynamic noise,among others. Additional examples including color drawings showingpseudocolor methods for displaying thermal imaging data are described inthe following U.S. provisional patent application, which is incorporatedherein by reference: Serial No. 60/256,852, filed Dec. 19, 2000.

EXAMPLE 1

[0100] This example describes a preferred sample holder for use inmeasurements of thermal processes, including chemical and physiologicalprocesses.

[0101]FIGS. 2 and 3 show a sample holder 200 constructed in accordancewith aspects of the invention. The sample holder includes a high thermalmass frame or base 202, a plurality of sample wells 204 and acorresponding plurality of windows 206, trapped volumes 208, referenceregions 209, and opaque coatings 210, and a cover 212.

[0102] Frame 202 is the main structural component of sample holder 200.The frame generally may be sized and shaped as desired, for bothconvenience and utility. Frame 202 is sized and shaped to form amicroplate, enabling the sample holder to be used with standardmicroplate equipment, such as handlers, washers, and/or readers, amongothers. A preferred frame is substantially rectangular, with a majordimension X of about 125-130 mm, a minor dimension Y of about 80-90 mm,and a height Z of about 5-15 mm, although other dimensions are possible.Frame 202 may include a base 214 configured to facilitate handlingand/or stacking, a notch 216 configured to facilitate receiving thecover, and/or a plurality of apertures 218 configured to receive andsupport a corresponding plurality of sample wells. The apertures provideclearance around the sample wells, creating an air gap that may providethermal isolation between the base and the sample well. The aperturesmay be formed using any suitable method, including machining and/orcasting the frame to include the apertures. The inner surface of eachaperture may be polished and/or lined with an opaque (i.e., lowtransmissivity) coating 210 such as AlSiO or gold to reflect infraredradiation and thus to form a thermal barrier to heat conduction to andfrom the sample wells. In this embodiment, adjacent sample wells may beseparated by two thermal barriers and a portion of the frame disposedbetween the two thermal barriers. The apertures and/or the sample wellsmay be tapered, such that the separation between the sample wells andthe walls of the corresponding apertures increases from the top to thebottom of the sample wells, further reducing conduction between thesample wells and the walls of the apertures.

[0103] The frame generally may be constructed of any suitable material.For example, frame 202 is constructed using a material having a highthermal mass (heat capacity) and high thermal conductivity, such asaluminum and/or other metals. Preferred materials such as aluminum mayreduce the time required for thermal stabilization within the testchamber while being sturdy enough for repeated, rugged use. Inparticular, a high thermal mass base (and/or an adjacent structure) mayfunction as a thermal buffer, helping to maintain a constant temperaturearound sample wells positioned in apertures 218.

[0104] Sample wells 204 are used to support and separate samples 220 forcalorimetric analysis. These sample wells may vary in size, shape,number, and arrangement, generally as desired, as long as the wells fitin the frame, and more particularly fit within the correspondingapertures in the frame. Exemplary sizes range between about 1 μL andabout 500 μL, and more preferably between about 1 μL and about 200 μL.Exemplary shapes include cones, frustums of cones, cylinders, andparallelepipeds, among others. Exemplary numbers include 96, 384, 864,1536, 3456, and 9600, among others. Exemplary arrangements includerectangular and hexagonal arrays, among others. Three preferredsample-well configurations that will fit as rectangular arrays within amicroplate-sized frame are listed in the following table: NumberArrangement Pitch (mm) Density (/mm²) of Wells of Wells Between Wells ofWells  96  8 × 12 9 1/81  384 16 × 24 4.5 4/81 1536 32 × 48 2.25 16/81 

[0105] Here, pitch is the center-to-center well-to-well spacing, anddensity is the number of wells per unit area. In a preferred embodiment,the frame will include a similarly spaced array of apertures forreceiving the sample wells. A preferred configuration includes 96frustoconical wells organized in an 8×12 rectangular array. Here,frustoconical refers to a well shape having conical sides and a flatbottom, as shown in FIG. 3.

[0106] The sample wells may be formed as cups that may be inserted intothe frame to hold a sample of calorimetric analysis, permitting ananalyzer to measure temperature changes in the sample, for example,resulting from chemical or physiological processes. In this way, asingle frame may be used to support cups of different sizes and shapes,so long as the cup (or cups) will fit within the correspondingapertures. The bottom surface 221 of the cup may be flat and/orparticularly thin in areas from which infrared measurements arecollected. A flat surface may serve to reduce optical distortion andcontrol reflections coming from the surface. A thin surface may enhanceinfrared transmission, because infrared properties generally areproportional to material thickness. A thin surface also may help toensure that the outer surface remains at or very close to thetemperature of the fluid.

[0107] The cup inserts may be formed individually or joined to form asheet or sheets of cups for use in the frame. Individual cups and smallsheets of cups generally provide greater flexibility, permitting cups tobe mixed and matched (e.g., according to size, shape, and/or infraredtransmission properties, among others) within a single plate. Largesheets of cups provide greater structural stability and convenience,permitting many cups to be changed at once. As mentioned above, cupinserts generally are configured so that a single cup resides within asingle aperture in the supporting frame. However, cup inserts also maybe configured so that two or more cups reside within a single aperture,permitting a single frame to support cups at two or more significantlydifferent sizes and/or densities.

[0108] The material properties of the cups are important for thermalisolation and infrared transmissivity. FIG. 4 shows the infraredtransmissivity of a preferred cup material as a function of materialthickness. This preferred material is an infrared-transmissive(polymeric) polyethylene blend sold under the trademark Poly IR 2™ byFresnel Technologies. The material has a high infrared transmissivitythat increases nonlinearly with decreasing material thickness, showing asignificant increase for thicknesses below about 0.001 inches. Moreover,the material has a low coefficient of thermal conductivity (˜0.6watts/meter-kelvin), which is about {fraction (1/10)} the thermalconductivity of most other infrared-transmitting materials, includingZn, Se, and Ge. In addition, the material has a low thermal mass, so itshould quickly assume the temperature of the sample. The low thermalmass of the cup combined with its low thermal conductivity andinsulation from the base reduce heat loss to the environment. Theinfrared transmission properties of the material allow the detector tomeasure the temperature of the fluid through the cup with less than 10%thermal contribution from the cup. Further aspects of the preferredsample well material are described in the following U.S. provisionalpatent application, which is incorporated herein by reference: SerialNo. 60/256,852, filed Dec. 19, 2000.

[0109] Window 206 is an environmental seal between the sample well andthe detector(located below the sample holder) when the sample holder isused with a bottom-read analyzer. The window may be formed of aninfrared-transmissive membrane material selected to enhance infraredtransmission within the spectral sensitivity band of the camera. Apreferred material is zinc selenide, which provides >97% transmission tothe bottom surface of the sample well.

[0110] Trapped volume 208 is formed between inner surfaces of frame 202,sample well 204, and window 206. The high thermal mass frame thatsurrounds the sample well acts as a capacitor to maintain a constanttemperature within the trapped volume, which typically contains air. Theinner surfaces of the frame may be lined with an opaque coating, asdescribed above.

[0111] Reference region 209 is a source of a reference signal for use inreference calibrations to reduce common-mode and parasitic noise, amongothers, as described above. Generally, each sample well includes ameasurement region and a corresponding reference region. The measurementregion generally comprises a portion of the sample or sample well, suchas an infrared transmissive bottom portion of the sample well for usewith bottom-read instruments. The reference region may comprise anadjacent portion of the frame, such as an annular donut-shaped portionformed around a perimeter and/or central axis of the measurement region.Here, the reference region is positioned at an end of a support memberformed by portions of the frame disposed between the sample wells. Thethermal mass of the thermal reference region and associated supportmember may be at least about the same as or greater than the thermalmass of the corresponding sample well and/or sample. The referenceregion may be formed of a high thermal mass and/or high (>0.8)emissivity material such as a metal that acts as an isolated, blackbodyreference. The thermal reference region may include a substantially flatemissive reference surface, where the emissive surface is substantiallyparallel to a flat portion of the bottom of the sample well and/or wherethe emissive surface is within a factor of ten of the area of a flatportion of the bottom of the sample well.

[0112] Cover 212 provides a mechanism for covering the sample holder, ora portion of the sample holder, to protect samples from evaporationand/or reduce the likelihood and amount of evaporation. Cover 212generally will leave a small air gap between the sample and the coverthat may saturate with fluid vapor to reduce evaporation. Cover includesan aperture 222 so that a fluid delivery system such as a pipette canpierce the cover and deliver reactant fluids.

EXAMPLE 2

[0113]FIG. 5 shows results from an experiment designed to measurethermal cross talk caused by heat conduction through the sample plate.The experiment was performed using a top-read thermal-imaging apparatusfitted with a quantum well (QWIP) infrared radiometer from FLIR Systems.The figure shows thermal images of two plates containing a fluid thatevaporates when in contact with dry ambient air. Here, relativetemperature is denoted by shading, where samples with relatively hightemperatures have increased shading, and samples with relatively lowtemperatures have reduced shading. Plate 1 (left) is fabricated using athin polymer insert and a high thermal mass base, as shown in FIGS. 2and 3. Plate 2 (right) is a standard commercially available 96-wellmicroplate fabricated from a polystyrene polymer (Costar 3628). Thethermal images show that plate 1 provides significantly better thermalisolation than plate 2. In particular, the wells in plate 1 areinsulated from the surrounding base material, reducing thermal “crosstalk” between adjacent wells, whereas the wells in plate 2 are poorlyinsulated, enhancing thermal “cross talk” and leading to significantthermal gradients across the plate.

EXAMPLE 3

[0114] This example describes results of an experiment designed to testthe effects of evaporation on the apparent temperature of samplespositioned in wells in a multiwell plate.

[0115] The experiment was performed using the top-read thermal-imagingapparatus and low cross-talk multiwell plate of Example 2. In theseexperiments, sets of adjacent wells were filled with fluids havingvarious evaporation characteristics or else were covered with ahigh-emissivity tape, as shown below. T T T T T T T T T T T T T T T W WW A A A T T T T T T W W W A A A T T T T T T W W W A A A T T T T T T O OO W W W T T T T T T O O O W W W T T T T T T O O O W W W T T T T T T T TT T T T T T T

[0116] Here, A=alcohol, W=water, O=mineral oil, and T=tape. Alcohol andwater are prone to evaporation, whereas mineral oil and tape are not.The apparent temperature in each well was measured at fixed intervalsduring a 15-minute period.

[0117] The data show that evaporation affects the apparent temperatureof the samples. Specifically, the apparent temperature of wellscontaining water or alcohol was about 27° C., whereas the apparenttemperature of wells containing mineral oil or tape was about 29° C., orabout 2° C. warmer. Apparently, evaporation of water and alcohol coolsthe layer of gas above these fluids, leading to lower measuredtemperatures.

[0118] The data also show that evaporation affects the apparenttemperature stability of the samples. Specifically, after compensatingfor common-mode noise, wells containing water or alcohol showed about0.1° C. peak-to-peak (PTP) temperature variations, whereas wellscontaining mineral oil or tape showed about 0.01° C. PTP temperaturevariations, or about one-tenth as large. Thus, evaporation may precludeaccurate measurement of small thermal processes within a well containinga fluid prone to evaporation, at least if measured from above the well.Conversely, reducing evaporation may improve thermal signal and permit amore accurate measurement of thermal reactions within the well.

EXAMPLE 4

[0119] This example describes results of an experiment designed todetermine whether temporal noise was caused by evaporation at thesurface of the fluid and whether temporal noise could be controlled byreading the fluid through a transparent film.

[0120] The experiment was performed using the top-read thermal-imagingapparatus and multiwell plate of Examples 2 and 3. However, here, eachwell was filled with water. Moreover, a first set of data was collectedas above, with the water exposed to ambient air, and a second set ofdata was collecting after placing a thin (˜0.0005-inch thick)infrared-transparent film (Poly IR II) over the wells, in direct contactwith the water to simulate a bottom-read design. The temperature in eachwell was again measured at fixed intervals during a 15-minute period.

[0121]FIG. 6A (“top read”) shows the range of measured temperatures as afunction of time, after subtraction of common-mode noise, reading fromthe surface of the exposed water. The data show significantthermodynamic noise, resulting from evaporation at the surface of thewater, with temperature variations of greater than about 0.1° C. (PTP).This noise level would make it very difficult to derive smalltemperature changes resulting from chemical or physiological processesbeneath the surface.

[0122]FIG. 6B (“bottom read”) shows the range of measured temperaturesas a function of time, after subtraction of common-mode noise, readingthrough the infrared-transparent film. The data show significantlyreduced temporal noise, with temperature variations of less than about0.025° C. (PTP). In this case, the film reduces or prevents evaporativecooling because the fluid no is longer exposed to dry air . Thisreduction in evaporative cooling reduces noise in the measurement, whichallows the system to record significantly smaller changes intemperature. This measurement technique, particularly when combined withthe novel multiwell plate design of FIGS. 2 and 3, allows accuraterecording of small subsurface processes taking place in the sample well,improving measurement resolution by about fourfold.

EXAMPLE 5

[0123]FIG. 7 shows results of an experiment designed to determine thepreferred number of frames to average in the frame-averagingnoise-reduction technique. The experiment was performed using top-readthermal-imaging apparatus and low cross-talk multiwell plate of Examples2-4. The experiments show the noise level at several areas of themultiwell plate, averaged over 0, 2, 4, 8, and 16 frames at 60 Hz. Thefollowing chart summarizes the data for selected areas within the image:Normal 2× Frames 4× Frames 8× Frames 16× Frames Max (bit 10656.0010632.00 10632.00 10616.00 10600.00 counts) Min (bit 10560.00 10576.0010584.00 10576.00 10568.00 counts) Avg (bit 10611.17 10604.05 10610.2810592.24 10582.40 counts) Range 0.60 0.35 0.30 0.25 0.20 (Kelvin) StdDev 0.10 0.07 0.05 0.04 0.04 (Kelvin)

[0124] Values are in 14 bit units. The “std dev” row is theroot-mean-squared (RMS) noise of a uniform target after application offrame-averaging.

EXAMPLE 6

[0125]FIG. 8 shows results of an experiment designed to determine thelevel of common-mode noise typical of an infrared camera. Theexperiments were performed using the apparatus and plate of Examples2-5. The figure shows a typical raw data set after frame-averaging(high-frequency noise reduction) and area averaging, but beforecommon-mode noise reduction and offset subtraction.

EXAMPLE 7

[0126]FIG. 9 shows results of an experiment designed to assess theability of offset subtraction to extract data for a 135-μW reaction in amultiwell plate. The experiments were performed using the apparatus andplate of Examples 2-6.

[0127]FIG. 9A shows raw data for an experiment using four sample wellsin which one well received a “sample” comprising a constant 135-μWinput, while the other wells received a benign (i.e., non-reactive)sample. The data show the average temperature in the measurement regionas a function of time, after image averaging is applied.

[0128]FIG. 9B shows the same data after offset subtraction, whichadjusts the data so that each measurement starts at zero at time zero.There is a residual common-mode noise of approximately 0.05° C. PTP,after offset subtraction. This common-mode noise may be out of phase andmay shift depending on the geometric position of the cell, as shown inFIG. 8.

EXAMPLE 8

[0129]FIG. 10 shows results of an experiment designed to assess theability of offset subtraction and the reduction of common-mode noiseusing a reference region local to the measurement region to extract datafor the 135-μW reaction of Example 7 and FIG. 9. The reduction ofcommon-mode noise reduces the noise level for the benign wells to an RMSlevel of about 0.004° C. The presence and thermal profile of the 135-μWreaction is clearly visible relative to the benign wells. The datacompare favorably to a thermal model that calculates the theoreticaleffect of a 135-μW input on a sample holder having the same fluidvolume. Without using the measurement and noise-reduction methodsdescribed here, the reaction resulting from a 135-μW input could not bedetected using an infrared camera.

EXAMPLE 9

[0130] This example describes software for performing and/or evaluatingcalorimetric measurements.

[0131]FIG. 11 shows a software screen for the display of thermal data.The software screen may include one or more data presentation fields.These fields may be used to present data using any suitable form,including tables, graphs, and pseudo-images, among others. The fieldsmay include a single display that includes or summarizes data frommultiple samples, and/or multiple displays that each include orsummarize data from one or a subset of the multiple samples. If thereare multiple displays, they may be arranged in a manner representativeof the layout of the corresponding samples, such as an 8×12 array ofmini-graphs corresponding to the 8×12 array of samples in a standard96-well microplate. The data displayed in the software screens mayinclude a characteristic of the thermal radiation detected, such as theamount, intensity, and/or spectrum of the radiation. The data also mayinclude a computed and/or processed quantity related to a characteristicof the thermal radiation detected, such as a temperature and/or a signalprocessed to reduce noise. The data also may include kinetic data, suchas temperature versus time (denoted “tick”). The software screen alsomay include software switches for selecting the scale of the display,for example, X-axis and Y-axis scales for graphical data and colorschemes for pseudocolor images, among others.

[0132]FIGS. 12 and 13 show software screens for collecting, displaying,and/or calibrating data relating to the measurement and referenceregions. The top screen shows the application screen for collecting datausing the circular reference around the perimeter of the measurementregion. The bottom screen shot shows the setup window for defining thecharacteristics of the reference. These screens again may include one ormore data presentation fields and one or more software switches, amongothers. Moreover, these screens may permit recording and/or reporting ofsystem parameters, such as emissivity, object distance, ambienttemperature, relative humidity, noise reduction methods, and/or sampleholder format, among others.

EXAMPLE 10

[0133] This example describes noise-reduction methods provided byaspects of the invention, particularly noise-reduction methods relatingto temporal and spatial noise.

[0134] The noise-reduction methods may include (1) converting detectedthermal infrared radiation to a signal, and (2) processing the signal toreduce the proportion of the signal that is attributable to noise. Thestep of processing the signal may include a step of (1) temporallyaveraging the signal comprising computing a quantity based ondistinguishable components of the signal representing thermal infraredradiation detected from the same sample at different times, and/or (2)replacing at least a portion of the signal with a revised portion formedfrom distinguishable components of the signal representing thermalinfrared radiation detected from the same position or positions in thesample at different times. Alternatively, or in addition, the step ofprocessing the signal may include a step of (1) spatially averaging thesignal comprising computing a quantity based on distinguishablecomponents of the signal representing thermal infrared radiationdetected from different portions of the same sample, and/or (2)replacing at least a portion of the signal with a revised portion formedfrom distinguishable components of the signal representing thermalinfrared radiation detected from the different positions in the sampleat the same time.

[0135] The time-based methods may be used to reduce temporal noise,among others, as described above in Section B. These methods may involvereplacing a portion of the signal with a revised portion formed for oneor more positions in one or more samples from distinguishable componentsof the signal representing thermal infrared radiation detected from atleast two, four, eight, or sixteen different times, among others. Thesemethods also may involve forming a weighted average of distinguishablecomponents of the signal representing thermal infrared radiationdetected from the same position in the sample at different times, forexample, by application of frame-averaging. In some embodiments, thesemethods may be adapted preferentially to reduce high-frequency temporalnoise in the signal, for example, by application of a low-pass temporalfilter and/or by replacing a relatively higher number of time datapoints with a relatively smaller number of time data points.

[0136] The space-based methods may be used analogously to reduce spatialnoise, among others, as described above in Section B. These methods mayinvolve replacing a portion of the signal with a revised portion formedat one or more different times from distinguishable components of thesignal representing thermal infrared radiation detected from at leasttwo, four, five, nine, or sixteen different positions in the sample,among others, and/or from different samples. These methods also mayinvolve forming a weighted average of distinguishable components of thesignal representing thermal infrared radiation detected from differentpositions in the sample at the same time. In some embodiments, thesemethods may be adapted preferentially to reduce high-frequency spatialnoise in the signal, for example, by application of a low-pass spatialfilter and/or by replacing a relatively higher number of spatial datapoints with a relatively smaller number of spatial data points.

[0137] The time-based and space-based methods may be used alone and/ortogether. Moreover, in many applications, both types of methods may beapplied more than once to a particular signal. For example, a signal maybe processed by replacing a portion of the signal with a revised portionformed from distinguishable components of the signal representingthermal infrared radiation detected from the same sample at differenttimes, and then re-processed by replacing at least a portion of therevised portion of the signal with a re-revised portion formed fromdistinguishable components of the revised portion representing thermalinfrared radiation detected from the same sample at different times.Here, the times used to process the signal and the times used tore-process the signal may be the same or different.

[0138] The noise-reduction methods also may include (1) detectingthermal infrared radiation transmitted from a reference region adjacenta sample, and (2) constructing a sample signal characteristic of thethermal infrared radiation detected from the sample based on the thermalinfrared radiation detected from the sample (e.g., as embodied in theprocessed signal) and the adjacent reference region. The referenceregion may comprise an annular portion of the sample plate distributedadjacent a perimeter and/or about a central or optical axis of thesample well.

[0139] The noise-reduction methods also may include repeating the stepsof detecting, converting, and/or processing for a plurality of samplesand/or reference regions. Thus, the methods may include (1) detectingthermal infrared radiation transmitted from a plurality of samplespositioned at a corresponding plurality of sample sites associated withthe sample holder, (2) converting the thermal infrared radiationdetected from each sample to a corresponding signal, and (3) processingeach signal to reduce noise, as described above. The thermal infraredradiation from each sample may be detected and/or processedsimultaneously and/or sequentially. The signals may be adjusted beforeand/or after processing so that each has the same preselected value atthe same preselected time. The preselected value may be zero, amongothers, and/or the preselected time may be zero, among others.

[0140] The disclosure set forth above encompasses multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious and directed to one of the inventions. These claims may referto “an” element or “a first” element or the equivalent thereof; suchclaims should be understood to include incorporation of one or more suchelements, neither requiring nor excluding two or more such elements.Inventions embodied in other combinations and subcombinations offeatures, functions, elements, and/or properties may be claimed throughamendment of the present claims or through presentation of new claims inthis or a related application. Such claims, whether directed to adifferent invention or to the same invention, and whether broader,narrower, equal, or different in scope to the original claims, also areregarded as included within the subject matter of the inventions of thepresent disclosure.

We claim:
 1. A method of detecting thermal infrared radiation,comprising: providing a sample substrate having a plurality of discretesample sites configured to support a corresponding plurality of samples;providing an optical device configured preferentially to detect thermalinfrared radiation; detecting thermal infrared radiation transmittedfrom a sample positioned at a corresponding sample site using theoptical device; converting the detected thermal infrared radiation to asignal; and processing the signal to reduce noise by replacing at leasta portion of the signal with a revised portion formed fromdistinguishable components of the signal representing thermal infraredradiation detected from the same position in the sample at differenttimes or from different positions in the sample at the same time.
 2. Themethod of claim 1, further comprising correlating the processed signalwith the progress of a chemical or physiological reaction occurring inthe sample.
 3. The method of claim 1, where the sample sites keep atleast a portion of each sample from mixing with at least a portion ofeach other sample.
 4. The method of claim 1, where the sample substrateis a microarray.
 5. The method of claim 1, where the sample sites keepeach portion of each sample from mixing with each portion of each othersample.
 6. The method of claim 1, where the sample substrate is amicroplate, and where the sample sites are microplate wells.
 7. Themethod of claim 1, where the number of sample sites is selected from thegroup consisting of 96, 384, 768, 1536, 3456, and
 9600. 8. The method ofclaim 1, where the density of sample sites is at least about 1 well per81 mm².
 9. The method of claim 1, where the optical device comprises: anexamination site; and a detector configured to receive andpreferentially to detect thermal infrared radiation transmitted from asample positioned at a sample site at the examination site.
 10. Themethod of claim 1, further comprising shielding the sample from incidentradiation to reduce the proportion of the signal arising fromtransmission, reflection, and/or photoluminescence from the sample. 11.The method of claim 1, further comprising filtering the radiationtransmitted from the sample to extract thermal infrared radiation priorto the step of detecting thermal infrared radiation.
 12. The method ofclaim 1, where at least about half of the thermal infrared radiationdetected by the optical device has a wavelength between about 3micrometers and about 5 micrometers.
 13. The method of claim 1, where atleast about half of the thermal infrared radiation detected by theoptical device has a wavelength between about 7 micrometers and about 14micrometers.
 14. The method of claim 1, where the processed signal isrepresentative of the temperature of the sample.
 15. The method of claim1, where the revised portion is formed from distinguishable componentsof the signal representing thermal infrared radiation detected from thesame position in the sample at different times and from differentpositions in the sample at the same time.
 16. The method of claim 1,where the revised portion is formed from distinguishable components ofthe signal representing thermal infrared radiation detected from thesame position in the sample at different times and not from differentpositions in the sample at the same time.
 17. The method of claim 1,where the revised portion is formed from distinguishable components ofthe signal representing thermal infrared radiation detected fromdifferent positions in the sample at the same time and not from the sameposition in the sample at different times.
 18. The method of claim 1,where the revised portion is formed for at least one position in thesample from distinguishable components of the signal from at least fourdifferent times.
 19. The method of claim 18, where the revised portionis formed for at least one position in the sample from distinguishablecomponents of the signal from at least sixteen different times.
 20. Themethod of claim 1, where the revised portion is formed for at least onetime from distinguishable components of the signal from at least fourdifferent positions in the sample.
 21. The method of claim 18, where therevised portion is formed for at least one time from distinguishablecomponents of the signal from at least nine different positions in thesample.
 22. The method of claim 1, where the step of processing isadapted preferentially to reduce high-frequency temporal noise in thesignal.
 23. The method of claim 1, where the step of processing isadapted preferentially to reduce high-frequency spatial noise in thesignal.
 24. The method of claim 1, where the step of processing thesignal includes the step of forming a weighted average ofdistinguishable components of the signal representing thermal infraredradiation detected from the same position in the sample at differenttimes.
 25. The method of claim 1, where the step of processing thesignal includes the step of forming a weighted average ofdistinguishable components of the signal representing thermal infraredradiation detected from different positions in the sample at the sametime.
 26. The method of claim 1, the revised portion being formed fromdistinguishable components of the signal representing thermal infraredradiation detected from the same sample at different times, furthercomprising re-processing the signal by replacing at least a portion ofthe revised portion of the signal with a re-revised portion formed fromdistinguishable components of the revised portion representing thermalinfrared radiation detected from the same sample at different times,where the times used to process the signal and the times used tore-process the signal may be the same or different.
 27. The method ofclaim 1, where the signal is an image, and where the step of processingthe signal includes forming a frame-average of a plurality of images.28. The method of claim 1, where the signal comprises a set of discretedata points, and where the step of processing the signal reduces thenumber of data points.
 29. The method of claim 1, further comprising:detecting thermal infrared radiation transmitted from a reference regionadjacent the sample site; and constructing a sample signalcharacteristic of the thermal infrared radiation detected from thesample based on the processed signal and the thermal infrared radiationdetected from the reference region.
 30. The method of claim 29, thesample sites having a central axis, where the thermal reference regionincludes an annular emissive reference surface positioned about thecentral axis of each sample site.
 31. The method of claim 29, where thethermal reference region has an emissivity of at least about 0.5. 32.The method of claim 29, further comprising repeating the steps ofdetecting thermal infrared radiation transmitted from a sample anddetecting thermal infrared radiation transmitted from an adjacentreference region for a plurality of samples positioned at acorresponding plurality of the sample sites associated with the sampleplate.
 33. The method of claim 1, further comprising covering the samplewells to reduce evaporative heat loss from the samples.
 34. The methodof claim 1, further comprising repeating the steps of detecting,converting, and processing for a plurality of samples positioned at acorresponding plurality of the sample sites associated with the samplesubstrate.
 35. The method of claim 34, where the thermal infraredradiation is detected simultaneously from the plurality of samples. 36.The method of claim 34, where the thermal infrared radiation is detectedsequentially from the plurality of samples.
 37. The method of claim 34,further comprising adjusting the processed signal corresponding to eachsample so that each processed signal has the same preselected value atthe same preselected time.
 38. The method of claim 37, where thepreselected value is zero.
 39. The method of claim 37, where thepreselected time is zero.
 40. The method of claim 34, further comprisingdisplaying the processed signals graphically in a manner representativeof the arrangement of the corresponding sample sites on the substrate.41. The method of claim 34, where the processed signal comprises asingle value for each sample at each time.
 42. A system for detectingthermal infrared radiation, comprising: a sample substrate having aplurality of discrete sample sites configured to support a correspondingplurality of samples; an optical device having an examination site and adetector, where the optical device is configured preferentially todetect thermal infrared radiation transmitted from a sample positionedat a corresponding sample site at the examination site; and a processorincorporating instructions for and capable of carrying out the functionof reducing noise by replacing at least a portion of the signal with arevised portion formed from distinguishable components of the signalrepresenting thermal infrared radiation detected from the same positionin the sample at different times or from different positions in thesample at the same time.
 43. A system for detecting thermal infraredradiation, comprising: means for supporting a plurality of samples at acorresponding plurality of discrete sample sites; means forpreferentially detecting thermal infrared radiation transmitted from asample positioned at a corresponding sample site; and means for reducingnoise by replacing at least a portion of the signal with a revisedportion formed from distinguishable components of the signalrepresenting thermal infrared radiation detected from the same positionin the sample at different times or from different positions in thesample at the same time.